MicroRNAs bind to sites in target mRNAs' 3'UTR and (principally) inhibit translation to protein. Since binding of microRNAs does not depend on full complementarity with their target sequences, microRNAs can bind to and block protein translation of many different mRNAs, and thereby serve as powerful regulatory switches. We profiled microRNA expression in hematopoietic stem-progenitor cells (HSPCs) and combined this data with human HSPC mRNA expression and thermodynamic microRNA-mRNA target predictions to propose that certain HSPC-expressed microRNAs (HE-microRNAs) regulate hematopoiesis. On this bioinformatic basis, we formulated a model for microRNA control of hematopoiesis in which many genes specifying hematopoietic differentiation are expressed by early HSPCs, but held in check by HE-microRNAs. For several target mRNAs important in hematopoiesis, we demonstrated experimentally that translation is actually decreased by HE- microRNAs. Mir-155 potently reduced both myelopoiesis and erythropoiesis of normal human HSPCs, and mir-16 selectively inhibited erythropoiesis. These findings supported our hypotheses, based on predicted target mRNAs, that (a) mir-155 may regulate development at approximately the common myeloid precursor (CMP) stage prior to erythroid commitment, while (b) mir-16 may block development at the stage of a bipotent megakaryocyte-erythroid progenitor (MEP). Thus, these 2 microRNAs may serve as tools to dissect the very early events of erythroid development and offer potential to expand these rare progenitors. A more detailed model of microRNA expression and molecular targets affecting erythropoiesis should provide additional molecular tools for studying erythroid development and function. Since the cells that we studied included rare adult stem cells and various stages of progenitors, we propose in Aim 1 to expand our microRNA profiles of HSPCs to more highly purified subsets of primary human and mouse erythroid progenitors. In addition, we will profile microRNA expression during erythroid development from NIH-approved human embryonic stem cells (hESCs). In Aim 2, we will determine if selected individual microRNAs experimentally inhibit development of erythroid progenitors. In Aim 3, we will study the molecular mechanisms of the microRNA's erythropoietic effects by identifying proteins whose synthesis is inhibited by each functionally-active microRNA. This project will investigate the effects of new regulatory molecules called microRNAs, which appear to play novel, potent roles in control of red blood cell formation. Understanding the actions of these microRNAs may provide new tools, for both expansion and control of red blood cell development.